Synthesis of MKNK1 in BL21 bacterial cells and purification by GST tag affinity chromatography
Primary Investigator (PI) Name
Carol Chrestensen
Department
CSM – Chemistry and Biochemistry
Abstract
Kinases are a class of enzymes that specialize in the transfer of a phosphate group to a target molecule, which is typically a protein. Kinases are critical in the regulation of various cellular processes, the activation or deactivation of specific enzymes, and cell signaling. MKNK1 is one such kinase, with three known isoforms of short, medium, and long lengths. MKNK 1 phosphorylates eukaryotic initiation factor 4E (eIF4E) through its interaction with eukaryotic initiation factor 4G (eIF4G), this phosphorylation alters protein translation but is not necessary for the process. Overexpression of MKNK 1 has been linked to several cancers, including stomach, endometrial, lung adenocarcinoma, and bladder cancers. Understanding how these isoforms function and interact with different substrates may provide insight into cancer-related signaling pathways and identify potential therapeutic targets. So far, DH5 alpha bacterial cells have been used to make copies of plasmid DNA, which encodes for the three isoforms of MKNK 1 with a GST tag. Gel electrophoresis analysis was then performed to ensure that the plasmid of interest was what was replicated in the bacterial cell. The same plasmid DNA was then transformed into BL21 cells for protein synthesis, and GST affinity chromatography was then used to purify MKNK 1 and its isoforms, respectively. To assess kinase activity, a PepTag assay was prepared using Kemptide, a synthetic peptide substrate known to be phosphorylated by MKNK 1 and PKA. The assay also requires ERK or p38 kinase-mediated phosphorylation. Our work intends to determine if these two kinases activate the three isoforms differently in vitro.
Disciplines
Biochemistry
Synthesis of MKNK1 in BL21 bacterial cells and purification by GST tag affinity chromatography
Kinases are a class of enzymes that specialize in the transfer of a phosphate group to a target molecule, which is typically a protein. Kinases are critical in the regulation of various cellular processes, the activation or deactivation of specific enzymes, and cell signaling. MKNK1 is one such kinase, with three known isoforms of short, medium, and long lengths. MKNK 1 phosphorylates eukaryotic initiation factor 4E (eIF4E) through its interaction with eukaryotic initiation factor 4G (eIF4G), this phosphorylation alters protein translation but is not necessary for the process. Overexpression of MKNK 1 has been linked to several cancers, including stomach, endometrial, lung adenocarcinoma, and bladder cancers. Understanding how these isoforms function and interact with different substrates may provide insight into cancer-related signaling pathways and identify potential therapeutic targets. So far, DH5 alpha bacterial cells have been used to make copies of plasmid DNA, which encodes for the three isoforms of MKNK 1 with a GST tag. Gel electrophoresis analysis was then performed to ensure that the plasmid of interest was what was replicated in the bacterial cell. The same plasmid DNA was then transformed into BL21 cells for protein synthesis, and GST affinity chromatography was then used to purify MKNK 1 and its isoforms, respectively. To assess kinase activity, a PepTag assay was prepared using Kemptide, a synthetic peptide substrate known to be phosphorylated by MKNK 1 and PKA. The assay also requires ERK or p38 kinase-mediated phosphorylation. Our work intends to determine if these two kinases activate the three isoforms differently in vitro.